PROJECT SUMMARY Mycobacterium tuberculosis (M.tb) is a major public health threat. M.tb replicates in macrophages by inhibiting phagosome-lysosome fusion. Lysosomal storage disorders (LSDs) including Gaucher's disease (GD) impair macrophage function, and a zebrafish model of GD has demonstrated increased susceptibility to Mycobacterium marinum. This suggests that human GD homozygous patients may be more susceptible to tuberculosis (TB). In contrast, it has been hypothesized that GD heterozygotes--as well as heterozygotes for other LSDs including Tay-Sachs Disease (TSD)--have a selective advantage of resistance to TB, and that this trait may be a key selective pressure for mutation retention in key populations. Using genome editing tools (TALENs and CRISPR/Cas9) we have generated hiPSCs from type 2 GD [homozygous for ?-glucocerebrosidase (GBA) 1448T>C mutation] patient-derived fibroblasts which are corrected for the GD mutation. The technology generates both biallelic (homozygous, GD +/+) and monoallelic (heterozygous, GD +/-) correction of the GBA mutation. These hiPSCs may be differentiated into macrophages to yield isogenic, GBA-corrected heterozygous (+/-) and homozygous (+/+) and uncorrected GD (-/-) cells for phenotypic studies. We propose to use these existing, novel, genome-engineered human stem cells to derive macrophages and test their phenotypic responses to M. tb infection. The goal of this proposal, then, is to test whether type 2 human GD -/- macrophages are more susceptible to M.tb infection than GD +/+ cells and whether heterozygosity (+/-) is associated with protection from TB using advanced cell infection phenotypic modeling. A mouse model of GD will be used to further evaluate the same questions in a non-human, mammalian model.